Method for site-specific polyvalent display on polymers

ABSTRACT

The present invention relates to novel complex peptidomimetic products comprising multiple homogeneous or heterogeneous pendant groups that are site-specifically positioned along a linear oligomer or polymer scaffold and methods of making thereof. More specifically, the invention relates to N-substituted glycine peptoid oligomers or peptoids and their use as substrates for azide-alkyne [3+2]-cycloaddition conjugation reactions and subsequent additional rounds of oligomerization and cycloaddition. The methods of the invention may also be used to generate peptoid-peptide hybrid or peptide products comprising multiple homogeneous or heterogeneous pendant groups, which are positioned precisely along the linear oligomer or polymer scaffold.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority under 35 USC §119(e) from U.S.Provisional Application Ser. No. 60/778,864, filed Mar. 3, 2006, whichapplication is herein specifically incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to novel complex peptidomimetic productscomprising multiple homogeneous or heterogeneous pendant groups that aresite-specifically positioned along a linear oligomer scaffold andmethods of making thereof. More specifically, the invention relates toN-substituted glycine peptoid oligomers or peptoids and their use assubstrates for azide-alkyne [3+2]-cycloaddition conjugation reactionsand subsequent additional rounds of oligomerization and cycloaddition.

BACKGROUND OF THE INVENTION

Several publications and patent documents are referenced in thisapplication in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of thesepublications and documents is incorporated by reference herein.

Techniques in bioconjugate chemistry have provided effective tools forendowing biomolecules with novel properties. Conjugation reactions areroutinely employed to modify proteins and nucleic acids so as toincorporate fluorophores, ligands, chelates, radioisotopes, affinitytags, and numerous other groups therein (G. T. Hermanson, BioconjugateTechniques, Academic Press: San Diego, Calif., 1996). When performed onsolid phase support, these reactions can be used to modify syntheticoligonucleotides and polypeptides with exceptional efficiency (reviewedin Virta et al., Tetrahedron, 2003, 59, 5137). In many cases,solid-phase conjugation reactions can be adapted for automatedprotocols, allowing the development of novel combinatorial libraries andmicroarray applications.

Polypeptides, while capable of exhibiting an extraordinary range ofbioactivities, often display poor pharmacological properties. For thisreason, synthetic mimics of peptides have been the focus of vigorousdevelopment by medicinal and bioorganic chemists. A variety ofoligomeric peptidomimetics have been introduced that show potential aspartial mimics of natural polypeptide species in that they exhibit someof the structural and functional attributes of natural polypeptides(Patch et al., Curr. Opin. Chem. Biol., 2002, 6, 872). Furtherelaboration of peptidomimetic structures may lead to a greater range ofcapabilities for this promising class of molecules.

N-substituted glycine oligomers (α-peptoids) and N-substituted β-alanineoligomers (β-peptoids) are examples of a promising class ofpeptidomimetics. Peptoids are oligomers based on a peptide backbone,which can be produced by an efficient, automated solid-phase synthesisthat facilitates the incorporation of diverse N-pendant sidechains in asequence-specific manner. As such, peptoids are a class of non-natural,sequence-specific polymers that represent an alternative derivative of apeptide backbone, the sequence and length of which can be preciselycontrolled. Structurally, peptoids differ from polypeptides in thattheir sidechains are pendant groups of the amide nitrogen rather thanthe α-carbon (Simon et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 9367;Zuckermann et al., J. Am. Chem. Soc., 1992, 114, 10646). Peptoids areparticularly useful for biomedical applications because these moleculesare largely invulnerable to protease degradation and hence are morestable than polypeptides in vivo. Peptoids have also been shown to bemore cell permeable than their peptide analogues (Kwon et al. J. Am.Chem. Soc., 2007, 129, 1508). These properties enhance bioavailability.Moreover, peptoids, which are synthetically produced by definition, canbe produced essentially in the absence of impurities.

Polyvalency is a powerful method utilized by nature to enhance thebinding strength of bioactive ligands. Polyvalency exerts its affect bymeans of the display of multiple copies of one or more chemical groups.Although these groups may possess only modest binding strengths inisolation, as part of a larger complex the binding interactions can sumto provide a very strong interaction. A variety of approaches have beendeveloped by chemists to mimic polyvalent display on polymers ordendrimers. Typically, these products exhibit limitations resulting fromthe difficulty of synthesis and/or the polydispersity of the scaffold.Ideally, chemists would seek to display ligands in a precise fashion inwhich the spacing between one or more of the conjugated groups can becontrolled. Thus, there is a need for novel methods that can be used toperform multi-site chemical conjugation onto an oligomer or polymerscaffold.

SUMMARY OF THE INVENTION

As described herein, the present inventors have developed a novel andprecise method for multi-site chemical conjugation onto an oligomer orpolymer scaffold. The present invention describes this method andpresents guidance as to how this method can be exploited to generatemolecules with therapeutic and/or diagnostic potential. Indeed, theinventors have successfully constructed linear oligomers comprisingeither multiple homogeneous or multiple heterogeneous pendant groupsusing the present method.

More specifically, the present method is directed to the generation of aseries of highly functionalized oligomers (e.g., peptoids) utilizing anovel sequential Cu(I) catalyzed azide-alkyne [3+2] cycloaddition(CuCAAC) reaction which is an example of, and herein referred to as, a“click chemistry” protocol. Accordingly, the present inventors havedeveloped an efficient method that enables the conjugation of a varietyof chemical moieties at precise locations along a peptidomimeticscaffold. The sequential CuCAAC reaction method introduced hereindemonstrates that 1,2,3-triazole linkages are compatible with multiplerounds of oligomer (e.g., peptoid) chain elongation on solid phasesupport. These techniques may prove suitable for similar sequentialbioconjugation of other polymers, including polypeptides, immobilized onsolid phase and may be amenable to automation. Moreover, the sequentialCuCAAC reaction method described herein may be used to generate hybridoligomers, such as, for example peptoid-peptide hybrids.

As described herein, the present inventors have utilized this approachto develop peptoids functionalized with groups appropriate forbiomedical applications, including moieties suitable for elaboration asconstituents of therapeutics, biosensors and molecular imaging agents.One such linear oligomer comprising homogeneous pendant groups is apeptoid which displays a plurality of identical hormone groups thattarget a particular hormone receptor implicated in, for example, breastor prostate cancer. Multivalent linear peptoids comprising hormonegroups may be used to advantage in therapeutic and/or diagnosticapplications. The method of the present invention may also be used togenerate a linear oligomer comprising heterogeneous pendant groups,wherein the linear oligomer is a peptoid which displays hormone groupsthat target a hormone receptor implicated in, for example, breast orprostate cancer and metal complexes that convey a therapeutic and/ordiagnostic functionality.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (Scheme 1) depicts multi-site modification of peptoid side-chainsby a sequential series of cycloaddition and oligomerization reactions.a) Coupling partner (0.06 M), CuI (0.11 M), ascorbic acid (0.06 M) andDIPEA (0.14 M) in DMF/pyridine (7/3 v/v), room temperature, 18 h. b) 95%TFA in H₂O, room temperature, 10 min.

FIG. 2 (Scheme 2) shows sequential click chemistry performed on solidphase support. All coupling partners were present at 0.06 M. a) CuI(0.11 M), ascorbic acid (0.06 M) and DIPEA (0.14 M) in2-butanol/DMF/pyridine (5/3/2 v/v/v), room temperature, 18 h. b) CuI(0.11 M), ascorbic acid (0.06 M) and DIPEA (0.14 M) in DMF/pyridine (7/3v/v), room temperature, 18 h. c) 95% TFA in H₂O, room temperature, 10min.

FIG. 3 depicts representative analytical RP-HPLC traces showing 6, 8, 10and 12 following cleavage from solid support. Chromatographic analysiswas performed on each of the crude products and is shown as overlaidtraces.

FIG. 4 shows the structure of a water-soluble bi-functionalized peptoidhexamer generated for biosensor applications.

FIG. 5 shows cyclic voltammetry curves of ethynylferrocene (0.5 mM) and13 (0.5 mM) in water with NaCl (50 mM) as supporting electrolyte, aglassy carbon working electrode, Ag/AgCl reference electrode and a Ptwire counter electrode with a scan rate at 9.0 mV s⁻¹.

FIG. 6 (Scheme 3) depicts a general method for synthesis of polyvalentselective hormone receptor cytotoxic agents. a) CuI (0.04 M), ascorbicacid (0.02 M) and DIPEA (0.04 M) in 2-butanol/DMF/pyridine (5/3/2v/v/v), rt, 18 h. b) CuI (0.11 M), ascorbic acid (0.06 M) and DIPEA(0.14 M) in DMF/pyridine (7/3 v/v), room temperature, 18 h. c) 95% TFAin H₂O, room temperature, 10 min.

FIG. 7 shows exemplary polyvalent selective hormone receptor cytotoxicagents. Bioactive ligands are displayed on a peptidomimetic scaffold viatriazole linkages. Compounds 1-3 show polyvalent display of17α-estradiol but are not limited to that specific hormone receptoragonist. Cytotoxic metallocenes are shown in mono-, bi- and trivalentdisplays.

FIG. 8 (Scheme 4) shows a general method for polyvalent display ofpeptides on a peptoid scaffold.

FIG. 9 (Scheme 5) shows a general method outlining the click chemistryprocedure to polyvalently display the azido peptides on a peptidomimeticscaffold.

FIG. 10 shows a graphical abstract of aspects of the present method.

FIG. 11 shows a postoligomerization modification protocol.

FIG. 12 shows postoligomerization modifications to pendant groups byregiospecific azide-alkyne [3+2] cycloaddition.

FIGS. 13A and B pictorially depict multivalent estradiol conjugates (A)and a hexavalent peptoid estradiol conjugate (B).

FIG. 14 shows a saturation curve of hormone binding in MCF-7 whole celllysates. MCF-7 cell extracts were prepared from logarithmically growingMCF-7 cells and hormone binding activity was measured as described inExample VI. Peptoid Estradiol conjugates: monovalent, divalent,trivalent, and hexavalent; E2: 17β-estradiol. For clarity, the insetshows binding activity of the trivalent conjugate only.

FIG. 15 shows a histogram of relative luciferase activity of lysatesderived from estrogen receptor positive (wtER) and estrogen receptornegative (Vo) human embryonic kidney (HEK) 293T cells. ER+(wtER) and ER−(Vo) HEK 293T cells were stably transfected with plasmids containingER-responsive luciferase reporter genes. Cells were treated for 18 hrswith 100 nM concentrations of the indicated ligand. Peptoid Estradiolconjugates: mono- (monovalent), di- (divalent), tri- (trivalent), andhexa- (hexavalent); E2: 17β-estradiol; Vehicle: EtOH.

FIG. 16 depicts chemical structures of several peptoid ethisteroneconjugates.

FIG. 17 depicts chemical structures of several estradiol conjugatesshowing extended alkyl linkers for enhanced receptor binding.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have focused on developing techniques forenhancing the structural complexity of a family of peptidomimetics knownas peptoids. In a particular embodiment, these oligomers are comprisedof N-substituted glycine monomer units and can exhibit a strongpropensity to form stable secondary structures (Kirshenbaum et al.,Proc. Natl. Acad. Sci. 1998, 95, 4303). Peptoids are efficientlysynthesized on solid phase support to incorporate a specific sequence ofchemically diverse monomer units (Zuckermann et al., J. Am. Chem. Soc.,1992, 114, 10646). Advances in molecular design approaches have enabledthe successful generation of peptoids that exhibit an array ofbiological activities (Patch et al., In Pseudopeptides in DrugDevelopment, Nielsen, P. E. Ed.; Wiley-VCH: Weinheim, Germany, 2004, 1).These products may prove to be well-suited for biomedical applicationsdue to their resistance to proteolytic degradation (Patch et al.,supra). As described herein, the goal of the present inventors is toenhance the structural and functional capabilities of peptoids bydeveloping new strategies for their chemical conjugation and ligation(Yoo et al., J. Am. Chem. Soc., 2005, 127, 17132).

The CuCAAC reaction is an example of a click chemistry reaction and isgaining prominence as a versatile technique for conjugating reactantsvia 1,2,3-triazole formation (Kolb et al., Drug Disc. Today, 2003, 8,1128). This example of a “click chemistry” reaction has been shown to beregiospecific and compatible with a wide range of substrates andreaction conditions. For example, azide-alkyne [3+2] cycloadditions havebeen employed to link polypeptide chains, synthesize dendrimers andconjugate derivatives to the exterior of viral particles (Franke et al.,Tetrahedron Lett., 2005, 46, 4479; Angelo et al., J. Am. Chem. Soc.,2005, 127, 17134; Joralemon et al., Macromolecules, 2005, 38, 5436; Wuet al., Chem. Comm., 2005, 46, 5775; Wang et al., J. Am. Chem. Soc.,2003, 125, 3192; Gupta et al., Bioconjugate Chem., 2005, 16, 1572). Thepresent inventors have demonstrated the advantages of using a clickchemistry approach for multi-site conjugation of alkyne- orazide-containing groups onto peptoid scaffolds (fang et al., Org. Lett.,2005, 7, 1951). Bioactive ligands typically contain chemicalfunctionalities that are incompatible with many solid phase synthesisprocedures. Due to the broad orthogonality of azide-alkyne [3+2]cycloaddition reactions, the present inventors were able to conjugatediverse ligands containing biologically relevant chemicalfunctionalities onto peptoid scaffolds with high efficiency. The netresult being a peptoid scaffold onto which a single type of biologicallyrelevant chemical functionality is multiply conjugated (i.e., conjugatedat multiple positions).

As described in detail herein below, the present inventors havedeveloped a novel procedure to enable the sequential conjugation ofmultiple, diverse groups onto a single oligomer scaffold throughsite-specific azide-alkyne [3+2] cycloaddition methods. In order togenerate peptoid substrates that allow for the consecutive addition ofheterogeneous pendant groups, a procedure was devised for modifyingreactive sidechain moieties and subsequently extending the oligomerscaffold. As reported herein, the technique developed utilizes asequential series of cycloaddition and scaffold extension reactions anddramatically enhances the functionality and chemical diversity ofpeptoid oligomers that can be generated. The ability to performsequential cycles of conjugation allows for the synthesis of complexmodular structures in which specific functionalities are displayed in asite-directed fashion. See Examples presented herein.

As described herein, the ability to synthesize linear oligomers to whichmultiple, heterogeneous chemical groups are precisely attached atpre-ordained positions offers the potential to be able to generatedesigner molecules that are multiply polyvalent. Suitablefunctionalities are known to those skilled in the art and include,without limitation, hormone receptor ligands, cell surface receptorligands, tumor specific antigen ligands, cytotoxic agents,pharmaceutical moieties, fluorophores, chelates, radioisotopes, andaffinity tags.

Various constituents may be incorporated into the oligomers of thepresent invention to improve cellular uptake or alter subcellularlocalization. Poly-Arg tails, for example, are known to promote cellularuptake. Skilled practitioners would be aware of other constituents thatmay be included in the oligomers of the present invention to improvetheir uptake in cells or alter subcellular localization.

With respect to hormone receptor ligands, several modified selectivehormone receptor modulators have gained prominence as potentialanti-tumor agents because they selectively modulate hormone receptoractivity in vitro. Current synthetic molecular design approaches for thepolyvalent display of selective hormone receptor agonists typicallyinvolve dendrimer scaffolds. Chemically modified hormone ligands arecovalently bound to dendrimer scaffolds that stem from a central core,resulting in polyvalent displays. While dendrimer-based agents haveproduced evidence of enhanced binding avidity, syntheses of thesemolecules usually require time-consuming and expensive multi-stepchemical ligation procedures. Moreover, dendrimer syntheses generallyproduce a heterogeneously dispersed product that renders isolation of apure product technically challenging, when possible. Yet anotherdrawback associated with dendrimers is that use of dendrimer-basedagents can lead to lysosomal storage disease. The present invention,therefore, offers substantial advantages with respect to both themethods involved in making dendrimers, which are time-consuming andexpensive, and the dendrimeric product generated, which may contributeto disease onset. The present method, therefore, offers a far moreefficient route with which to generate polyvalent molecules and producesa polyvalent linear product that is both structurally and functionallydistinct from that of a dendrimer to which the same polyvalent groupshave been attached.

As described herein, a polyvalent oligomer comprising a plurality ofselective hormone receptor ligand conjugates may be used as an exemplarypolyvalent selective hormone receptor agent. These molecules incorporatemultiple hormone receptor ligands onto a single peptidomimetic scaffold.As such, these molecules are linear oligomers comprising homogeneouspendant groups, wherein the linear oligomer comprises monomers andmultiple homogeneous pendant groups are attached directly to thebackbone of the linear oligomer. For the synthesis of these agents,N-substituted glycine oligomer (α peptoid) scaffolds that includemultiple reactive sites are generated through solid phase synthesisprotocols. 17α-ethynylestradiol is ligated to the oligomer scaffoldthrough highly regiospecific Cu catalyzed azide-alkyne [3+2]cycloaddition reactions resulting in 1,2,3 triazole linkages between thescaffold and the 17α-ethynylestradiol moieties. This rapid and efficientprocedure can be used to generate peptidomimetic scaffolds that comprisepolyvalently displayed hormone receptor ligands. See Examples IV and VI.

With respect to metallocenes, these are organo-metallic species in whicha metal atom is sandwiched between two aromatic ligands. An exemplarymetallocene is ferrocene, in which an iron atom is bound between twocyclopentadiene groups. Metallocenes in general, and ferrocenes inparticular, are of interest for pharmaceutical and biosensorapplications due to their redox activity. Ferrocenes, for example, canexhibit a potent cytotoxic effect primarily due to oxidative damage theycause to DNA.

Metallocene derivatives of selective hormone receptor agonists arecurrently being developed and their antiproliferative effects are beingtested in a number of cancer cell lines. Although these agents aredesigned to include hormone agonists and cytotoxic agents within thesame molecule, binding strengths can be low due to the monovalentdisplay of the receptor ligand. The present invention addresses thisdeficiency of the available molecules by achieving polyvalent display ofhormone receptor ligands and cytotoxic agents on a single scaffold.

As described herein, a polyvalent oligomer comprising at least oneselective hormone receptor ligand conjugate and at least one metalloceneconjugate may be used as an exemplary polyvalent selective hormonereceptor cytotoxic agent. These molecules are designed to incorporatemultiple hormone receptor ligands and cytotoxic moieties onto a singlepeptidomimetic scaffold. For the synthesis of these agents,N-substituted glycine oligomer (peptoid) scaffolds including multiplereactive sites are generated through solid phase synthesis protocols.Cytotoxic metallocenes (e.g., ethynylferrocene) are ligated to theoligomer scaffold through highly regiospecific azide-alkyne [3+2]cycloaddition reactions resulting in 1,2,3 triazole linkages between thescaffold and the ethynylferrocene moieties. The oligomer chain length isthen extended to include additional azide functionalities.17α-ethynylestradiol is then ligated to the oligomer scaffold resultingin polyvalent display of the hormone receptor agonist (FIG. 6; Scheme3). This rapid and efficient procedure can be used to generatepeptidomimetic scaffolds that comprise polyvalently displayed hormonereceptor ligands and cytotoxic agents (FIG. 7, Compounds 1-3).

In addition, polyvalent displays can be constructed by conjugation tomacromolecular peptoid scaffolds. The term macromolecular oligomerscaffold refers to peptoids of very long chain length, up to andpotentially in excess of 1,000 submonomers, which may be generated usingpreviously described ligation procedures (Yoo et al. J. Am. Chem. Soc.2005, 127, 17132). These scaffolds can be synthesized by theprotease-mediated ligation of individual peptoid oligomers. The productswill allow polyvalent display of from tens to hundreds of copies of theconjugated species.

It is noteworthy that conjugating bioactive ligands to peptidomimeticscaffolds via Cu(I) catalyzed azide alkyne [3+2] cycloaddition (clickchemistry) demonstrates several advantages over incorporating themoieties directly as submonomers. The click chemistry method facilitatesdirect conjugation of commercially available azide or alkyne containingbioactive ligands to the peptoid scaffolds of the present inventionwithout the need for further chemical modification. Additionally, manybioactive ligands contain chemical functionalities that are incompatiblewith many solid phase synthesis procedures. By exploiting the clickchemistry method to conjugate functionalities onto a peptidomimeticscaffold, the present inventors have been able to polyvalently displaydiverse, bioactive ligands that would otherwise be extremely difficultor impossible to incorporate using the submonomer method.

Moreover, the versatility of the present method is underscored by theability to conjugate bioactive ligands to peptidomimetic scaffoldsthrough linkers that separate the moieties from the scaffold backbone.When binding to receptor molecules or enzymes, bioactive ligandstypically become fully sequestered within the active site of theproteins. Inclusion of a spacer (i.e., a linker) between the ligand andthe scaffold may enhance the overall therapeutic effect of moleculesproduced using the present method by allowing the bioactive moieties tobe fully incorporated into the active site. Thus, the click chemistrymethod conveys the freedom to control the spacing between the scaffoldand the bioactive ligand, thereby limiting issues relating to sterichindrance between the bioactive ligand and the scaffold and/or otherconjugated moieties. As demonstrated herein, the present inventors havebeen able to extend the linker between the triazole linkage and theligand itself (See FIG. 17), with the intent that such spacing mayenhance binding properties. Incorporating the moieties directly assubmonomers limits this ability.

While the present inventors have shown that the click chemistryprocedure described herein can be used to conjugate multiple bioactiveligands to peptoid scaffolds with high efficiency, improvements in thebasic procedure are offered herein to overcome certain limitations thatmay arise in the context of some bioactive pendant groups. Using thebasic procedure, it is necessary to wait until the last round of clickchemistry to conjugate bioactive pendant groups that contain chemicalfunctionalities that are incompatible with peptoid extension methods,such as, for example, 17α-ethynylestradiol. By modifying the sequentialclick chemistry method to include a step directed to protecting theincompatible chemical functionalities, greater flexibility as to thepositioning of pendant group attachment sites in the oligomer sequencecan be achieved.

As described herein, the method of the present invention may be used togenerate cyclic oligomers comprising homogeneous or heterogeneouspendant groups. In accordance with the present method, cyclic peptoids,for example, may be generated by synthesizing a linear peptoid, cleavingit from solid phase, cyclizing the linear peptoid, and conjugating it topendant groups at multiple sites. Cyclic oligomers may also be generatedby synthesizing linear oligomers comprising homogeneous or heterogeneouspendant groups which are subsequently circularized. Cyclic oligomers(e.g., cyclic peptoids) may be used to particular advantage in thecontext of engaging cell surface receptor clusters. For a reviewdetailing the use of cyclic oligomers as ligands for multivalentreceptors, see Kiessling et al., Angew Chem Int Ed Engl., 2006, 45,2348, which is incorporated herein by reference in its entirety.

A skilled practitioner would appreciate that the method of the presentinvention may be performed in either solid phase or solution phase, orcombinations thereof, depending on the desired outcome of the synthesisprotocol.

The following is a non-limiting list of protecting groups that may beused in the context of the present invention. Alcohol protecting groupsinclude, for example, Acetyl (Ac), which can be removed by acid or base;Tetrahydropyranyl ether (THP), which can be removed by acid;Methoxymethyl ether (MOM), which can be removed by acid;β-Methoxyethoxymethyl ether (MEM), which can be removed by acid;p-Methoxybenzyl ether (PMB), which can be removed by acid orhydrogenolysis; and Methylthiomethyl ether, which can be removed byacid. Amine protecting groups include, for example, Carbobenzyloxy group(CBZ), which can be removed by hydrogenolysis; Tert-butyloxycarbonyl(BOC) group, which can be removed by concentrated, strong acid (such asHCl or CF₃COOH); 9-Fluorenylmethyloxycarbonyl group (FMOC), which can beremoved by base; and Benzyl (Bn), which can be removed byhydrogenolysis. Carbonyl protecting groups include, for example,Acetals, which can be removed by acid. Carboxylic acid protecting groupsinclude, for example, Methyl esters, which can be removed by acid orbase; and Benzyl esters, which can be removed by hydrogenolysis. Askilled practitioner would be aware of additional protecting groups ofutility with respect to different pendant groups and would be able toselect an alternative protecting group or groups based on his/herexperience and the desired polyvalent peptoid comprising heterogeneouspendant groups to be generated.

In order to more clearly set forth the parameters of the presentinvention, the following definitions are used:

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, reference to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

The terms “peptoid” and “polypeptoid” encompass both α- and β-peptoids.The following terms may be used interchangeably herein with α-peptoids,“poly (N-substituted glycines)”, “oligo (N-substituted) glycines”, and“oligomeric N-substituted glycines”. “Peptoids” and “polypeptoids” maybe produced using the methodology of the present invention. As indicatedherein, peptoids are not peptides in that they are not composed ofnaturally-occurring amino acids linked by peptide bonds. Peptoids may,however, be designed to possess features (e.g., reactive sites) thatstructurally mimic naturally occurring peptides and proteins, and assuch are useful as potential therapeutic agents and/or as detectionreagents. The term “α-peptoid” refers to a plurality of oligomericN-substituted glycines of any length. The term “β-peptoid” refers to aplurality of oligomeric N-substituted β-alanines of any length. Morespecifically, a peptoid of the invention is between 2-1,000 monomers,more particularly between 2-100 or 2-25.

Peptoids can be synthesized in a sequence-specific fashion using anautomated solid-phase protocol, e.g., the sub-monomer synthetic route.See, for example, Wallace et al., Adv. Amino Acid MimeticsPeptidomimetics, 1999, 2, 1-51 and references cited therein and Patch etal., In Pseudopeptides in Drug Development, Nielsen, ed., Wiley-VCH,Weinheim, Germany, 2004, p. 1, all of which are incorporated herein intheir entirety by this reference. The synthesis of macromolecularpeptoids can be achieved using the ligation protocol as described by Yooet al. (supra) and such peptoids may comprise alternating methoxy andbenzyl side chains.

As indicated above, α-peptoids (N-substituted glycines) are non-natural,sequence specific polymers composed of a poly-glycine backbone, whereasβ-peptoids (N-substituted β-alanines) are non-natural, sequence specificpolymers composed of a poly-alanine backbone. Peptoid molecules containfunctional groups (R) positioned as substituents of the amide nitrogen.A linear peptoid oligomer is distinct from that of a branched peptoidoligomer with respect to the nature of the characteristic “R” group.Linear peptoids contain R groups that do not include peptoid oligomersthemselves. In contrast, branched peptoids are peptoids that comprise atleast one R group that is a peptoid oligomer. Basic structuresillustrating the structural differences exhibited by different types ofpeptoids are presented below.

Linear Peptoid:

A “substrate” or “solid support” is a conventional solid supportmaterial used in peptide synthesis. Non-limiting examples of suchsubstrates or supports include a variety of solid supports, includingRink Amide resin, Wang resin and Chlorotrityl resin. Connectors to thesolid supports such as those which are photocleavable, DKP-forminglinkers (DKP is diketopiperazine; see, e.g., WO90 09395 incorporatedherein by reference), TFA cleavable, HF cleavable, fluoride ioncleavable, reductively cleavable and base-labile linkers are alsoencompassed herein. A solid support may also comprise a plurality ofsolid support particles, such as beads, which can be split into portionsor “subamounts” for separate reactions and recombined as desired.

As used herein, the terms “immobilized on solid phase” or “solidsupport-bound” refer to molecules that are attached to a solid phase orsolid support. Such attachments may be reversible in nature. A skilledpractitioner is familiar with a variety of reversible attachment modesand various protocols to effect release of immobilized molecules fromsolid supports to which they are attached.

As described herein, the polyvalent linear and/or cyclized peptoidscomprising homogeneous or heterogeneous pendant groups of the inventionmay be used as therapeutic and/or diagnostic molecules. Their use aspotential vaccines is also encompassed herein. Polyvalent linear and/orcyclized peptoids which are glycoconjugates are also envisioned for avariety of applications. With respect to diagnostic agents, thepolyvalent linear and/or cyclized peptoids comprising homogeneous orheterogeneous pendant groups of the invention may be used to advantageas molecular imaging agents, such as, without limitation, magneticresonance imaging (MRI) contrast agents, positron emission tomography(PET) imaging agents, and optical imaging agents.

The invention also includes a composition for diagnosis or therapycomprising an effective amount of a polyvalent linear peptoid comprisinghomogeneous or heterogeneous pendant groups of the invention and aphysiologically acceptable excipient or carrier.

Physiologically acceptable and pharmaceutically acceptable excipientsand carriers for use with peptoid type reagents are well known to thoseof skill in the art.

By “physiologically or pharmaceutically acceptable carrier” as usedherein is meant any substantially non-toxic carrier for administrationin which the peptoids of the invention are stable and bioavailable whenused. The peptoid can, for example, be dissolved in a liquid, dispersedor emulsified in a medium in a conventional manner to form a liquidpreparation or is mixed with a semi-solid (gel) or solid carrier to forma paste, ointment, cream, lotion or the like.

Suitable carriers include water, petroleum jelly (vaseline), petrolatum,mineral oil, vegetable oil, animal oil, organic and inorganic waxes,such as microcrystalline, paraffin and ozocerite wax, natural polymers,such as xanthanes, gelatin, cellulose, or gum arabic, syntheticpolymers, such as discussed below, alcohols, polyols, water and thelike. Preferably, because of its non-toxic properties, the carrier is awater miscible carrier composition that is substantially miscible inwater. Water miscible carrier compositions can include those made withone or more ingredients set forth above but can also include sustainedor delayed release carrier, including water containing, waterdispersable or water soluble compositions, such as liposomes,microsponges, microspheres or microcapsules, aqueous base ointments,water-in-oil or oil-in-water emulsions or gels.

In one embodiment of the invention, the carrier comprises a sustainedrelease or delayed release carrier. The carrier is any material capableof sustained or delayed release of the peptoid to provide a moreefficient administration resulting in one or more of less frequentand/or decreased dosage of the peptoid, ease of handling, and extendedor delayed effects. The carrier is capable of releasing the oligomerwhen exposed to the environment of the area for diagnosis or treatmentor by diffusing or by release dependent on the degree of loading of thepeptoid to the carrier in order to obtain peptoid release. Non-limitingexamples of such carriers include liposomes, microsponges, microspheres,or microcapsules of natural and synthetic polymers and the like.Examples of suitable carriers for sustained or delayed release in amoist environment include gelatin, gum arabic, xanthane polymers; bydegree of loading include lignin polymers and the like; by oily, fattyor waxy environment include thermoplastic or flexible thermoset resin orelastomer including thermoplastic resins such as polyvinyl halides,polyvinyl esters, polyvinylidene halides and halogenated polyolefins,elastomers such as brasiliensis, polydienes, and halogenated natural andsynthetic rubbers, and flexible thermoset resins such as polyurethanes,epoxy resins and the like.

Preferably, the sustained or delayed release carrier is a liposome,microsponge, microphere or gel.

The compositions of the invention are administered by any suitablemeans, including injection, transdermal, intraocular, transmucosal,bucal, intrapulmonary, and oral. While not required, it is desirablethat parenteral compositions maintain the peptoid at the desiredlocation for about 24 to 48 hours; thus, sustained release formulationscan be used, including injectable and implantable formulations.

If desired, one or more additional ingredients can be combined in thecarrier: such as a moisturizer, vitamins, emulsifier, dispersing agent,wetting agent, odor-modifying agent, gelling agents, stabilizer,propellant, antimicrobial agents, sunscreen, and the like. Those ofskill in the art of diagnostic pharmaceutical formulations can readilyselect the appropriate specific additional ingredients and amountsthereof. Suitable non-limiting examples of additional ingredientsinclude stearyl alcohol, isopropyl myristate, sorbitan monooleate,polyoxyethylene stearate, propylene glycol, water, alkali or alkalineearth lauryl sulfate, methylparaben, octyl dimethyl-p-amino benzoic acid(Padimate O), uric acid, reticulan, polymucosaccharides, hyaluronicacids, aloe vera, lecithin, polyoxyethylene sorbitan monooleate,tocopherol (Vitamin E) or the like.

More particularly, the carrier is a pH balanced buffered aqueoussolution for injection. The particular carrier used, however, will varywith the mode of administration.

The compositions for administration usually contain from about 0.0001%to about 90% by weight of the peptoid compared to the total weight ofthe composition, more particularly from about 0.5% to about 20% byweight of the peptoid compared to the total composition, and even moreparticularly from about 2% to about 20% by weight of the peptoidcompared to the total composition.

The effective amount of the peptoid used for therapy or diagnosis willvary depending on one or more factors such as the specific peptoid used,the age and weight of the patient, the type of formulation and carrieringredients, frequency of use, the type of therapy or diagnosispreformed and the like. It is a matter of routine for a skilledpractitioner to determine the precise amounts to use, taking intoconsideration these factors and the present specification.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, preferred methods and materialsare described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features andadvantages of the invention will be apparent from the detaileddescription, examples, and the claims.

Example I Methods and Materials

General:

Peptoid oligomers were characterized by analytical Reversed-Phase HighPerformance Liquid Chromatography (RP-HPLC) using a C4 column (PeekeScientific, Ultra-120, 5 μm, 120 Å, 2.0×50 mm) on a Beckman CoulterSystem Gold HPLC system. Products were detected by UV absorbance at 214nm with a System Gold 166 detector. Data were analyzed with BeckmanCoulter 32 Karat software version 5.0. Unless otherwise noted, lineargradients were conducted from 5% to 95% solvent B (0.1% TFA in HPLCgrade acetonitrile) over solvent A (0.1% TFA in HPLC grade water) in 10minutes with a flow rate of 0.7 mL min⁻¹. Subsequent chain elongationand average coupling yields were estimated by RP-HPLC using methodsdescribed previously by Jang et al. (supra).

Additional characterization of peptoid oligomers was conducted usingLiquid Chromatography/Mass Spectrometry (LC/MS). All peptoids describedherein were analyzed using an Agilent 1100 Series LC/MSD Trap XCTequipped with an electrospray ion source. All LC/MS experiments werepreformed in positive ion mode. Unless otherwise stated, all analyseswere performed on peptoids cleaved from resin without furtherpurification.

Peptoid Synthesis:

Synthesis of peptoid oligomers was conducted on Rink Amide resin(Novabiochem, San Diego, Calif.), using a modification of the standardsubmonomer synthesis procedures described by Zuckermann et al. (supra).Peptoids were synthesized by manual techniques as well as automatedprocedures on a robotic workstation (Charybdis Instruments) withsoftware program files written in house. All reactions were conducted onsolid-phase at room temperature.

Typically, 100 mg of Rink Amide resin at a loading level of 0.55 mmolg⁻¹ was swollen in 3 mL of dichloromethane (DCM) for 45 minutes beforeFmoc deprotection. Multiple washing steps using N,N′-dimethylformamide(DMF) (4×2 mL) and DCM (3×2 mL) were performed between each syntheticprocedure described below. All reactant equivalents are based on resinloading level for a given amount of resin. Resin was Fmoc deprotected bytreatment with 20% piperidine in DMF (15 mL g⁻¹ resin, 20 minutes).Deprotection reagents were washed from the resin and approximately 20stoichiometric equivalents (eq) bromoacetic acid (1.2 M in DMF, 8.5 mLg⁻¹ resin) and 24 eq diisopropylcarbodiimide (2 mL g⁻¹ resin) wereadded. The bromoacetylation reaction mixture was agitated at roomtemperature for 20 minutes. Following washing, 20 eq monomer amine (1.0M in DMF, 10 mL g⁻¹ resin) were added and the reaction was agitated for20 minutes. Bromoacetylations and monomer amine displacements wererepeated until peptoid oligomers of desired length were achieved.

Peptoid products were cleaved from solid support by treatment with 95%trifluoroacetic acid (TFA) in water (40 mL g⁻¹ resin) for 10 minutes.The TFA cleavage cocktail was evaporated under nitrogen. Forcharacterization by RP-HPLC and LC/MS, peptoids were resuspended in 1 mLHPLC solvent (50% acetonitrile in water).

Polyfunctionalized Peptoid 24-mer (4):

Linear peptoid dodecamers were synthesized with high efficiency usingtechniques described above. Compound 1 was allowed to react with 21 eqphenyl propargyl ether (0.06 M), 40 eq CuI (0.11 M), 20 eq ascorbic acid(0.06 M) and 50 eq DIPEA (0.14 M) in 20 mL DMF/pyridine 7/3 v/v (0.2 mLme resin) in a 20 mL scintillation vial (Wheaton Scientific, Millville,N.J.), generating compound 2. In order to completely dissolve the solidreactants, the vial was placed in a bath sonicator (VWR Aquasonic 75HT)and sonicated for 5-10 minutes. The vial was purged with gaseousnitrogen, tightly capped, sealed with Parafilm and shaken at roomtemperature for 18 hours. Following completion of the reaction, theresin was transferred to a 10 mL fitted syringe (Torviq) and washed withDMF (7×3 mL), Cu scavenger cocktail (DMF/pyridine 6/5 v/v, ascorbic acid0.02 g mL⁻¹) (7×3 mL) and DCM (7×3 mL). The resin was then dried undernitrogen gas flow and approximately 3 mg was removed forcharacterization. Peptoid 2 was then elongated to a 24-mer (FIG. 1,Scheme 1, Compound 3) through twelve rounds of monomer amine addition asdescribed above. Compound 3 was added to 21 eq benzyl azide (0.06 M), 40eq CuI (0.11 M), 20 eq ascorbic acid (0.06 M) and 50 eq DIPEA (0.14 M)in 20 mL DMF/pyridine 7/3 v/v (0.2 mL me resin) in a 20 mL scintillationvial. The vial was purged with gaseous nitrogen, tightly capped, sealedwith Parafilm and was allowed to stir at room temperature for 18 hours,affording peptoid 4. Following washing steps, the generation of peptoid4 was confirmed using RP-HPLC and LC/MS. Overall purity of peptoid 4 wasfound to be >35% as calculated by analytical RP-HPLC. Sequencing ofpeptoid 4 was conducted using a MS/MS fragmentation technique on anAgilent 1100 Series LC/MSD Trap XCT equipped with an electrospray ionsource. LC/MS² experiments were performed in positive ion mode.

Synthesis of Multi-Functionalized Peptoid Dodecamer (12):

Approximately 100 mg of peptoid 5-bound Rink Amide resin was swollen inDCM for 45 minutes. The DCM was removed and the swollen resin wastransferred to a 20 mL scintillation vial. Depending on thehydrophobicity of the respective coupling partner, the resin wassuspended in either 20 mL 2-butanol/DMF/pyridine 5/3/2 v/v/v (0.2 mLmg⁻¹ resin) or 20 mL DMF/pyridine 7/3 v/v (0.2 mL mg⁻¹ resin). Tocompound 5 was added 21 eq phenyl propargyl ether (0.06 M) along with 40eq CuI (0.11 M), 20 eq ascorbic acid (0.06 M) and 50 eq DIPEA (0.14 M)to generate peptoid 6. In order to completely dissolve the solidreactants, the vial was placed in a bath sonicator (VWR Aquasonic 75HT)and sonicated for 5-10 minutes. The vial was purged with nitrogen,tightly capped, sealed with Parafilm and vigorously shaken at roomtemperature for 18 hours. Following completion of the reaction, theresin was transferred to a 10 mL fritted syringe (Torviq) and washedwith DMF (7×3 mL), Cu scavenger cocktail (DMF/pyridine 6/5 v/v, ascorbicacid 0.02 g mL⁻¹) (7×3 mL), and DCM (7×3 mL). The resin was then driedunder nitrogen gas flow and approximately 3 mg was removed forcharacterization.

Resin-bound peptoid trimer 6 was elongated to a hexamer using techniquesas described (FIG. 2; Scheme 2, Compound 7). Benzyl azide (0.06 M) wasthen coupled onto the terminal alkyne functionality of 7 using methodsoutlined above, generating compound 8. The click chemistry reagents werewashed from the resin and a small amount of compound (3 mg) was removedfor characterization. This sequential click chemistry reaction methodwas repeated until four complete rounds of elongation and cycloadditionhad been achieved (FIG. 2; Scheme 2, Compound 12). Compound 12 wasconfirmed using RP-HPLC (FIG. 3) and MS/MS sequencing techniques.

Synthesis of Peptoid-Ferrocene Conjugate (13):

Approximately 100 mg of Rink

Amide resin was swollen in DCM for 45 minutes. The DCM was removed andthe resin was Fmoc deprotected by treating it with 20% piperidine in DMF(15 mL g⁻¹ resin, 20 min). Deprotection reagents were washed from theresin and peptoid trimers were generated that contained twomethoxyethylamine monomers and a terminal azidopropyl moiety. The resinwas transferred to a 20 mL scintillation vial and ethynylferrocene (21eq, 0.06 M), 40 eq CuI (0.11 M), 20 eq ascorbic acid (0.06 M) and 50 eqDIPEA (0.14 M) were added to the resin-bound peptoid in 20 mL2-butanol/DMF/pyridine 5/3/2 v/v/v (0.2 mL mg⁻¹ resin). The vial waspurged with nitrogen gas, sealed with Parafilm and shaken at roomtemperature for 18 hours. The click chemistry reagents were washed fromthe resin and a small amount of the peptoid-ferrocene conjugate wasremoved for characterization. This monofunctionalized peptoid trimer wasthen elongated to a hexamer with two additional methoxyethylaminemonomers and a terminal azidopropyl moiety (Compound 14). The resin wastransferred to a 20 mL scintillation vial and 17α-ethynylestradiol (21eq, 0.06 M), 40 eq CuI (0.11 M), 20 eq ascorbic acid (0.06 M) and 50 eqDIPEA (0.14 M) were added to the resin-bound peptoid in 20 mLDMF/pyridine 7/3 v/v mL (0.2 mL mg⁻¹ resin). The vial was purged withnitrogen, tightly capped, sealed with Parafilm and shaken at roomtemperature for 18 hours. Following completion of the reaction, theresin was transferred to a 10 mL fritted syringe (Torviq) and washedwith DMF (7×3 mL), Cu scavenger cocktail (DMF/pyridine 6/5 v/v, ascorbicacid 0.02 g mL⁻¹) (7×3 mL) and DCM (7×3 mL). The resin was then driedunder nitrogen gas flow and approximately 3 mg was removed forcharacterization. Peptoid 13 was purified to >96% purity as calculatedby RP-HPLC.

Electrochemical Analyses:

Ethynylferrocene (1.05 mg, 5.0 μmol) was dissolved in 10 mL HPLC gradewater. NaCl (29.22 mg, 0.5 mmol) was added to the solution as asupporting electrolyte. Similarly, solutions of 10 mL water, 6.3 mg (5.0peptoid 13 or 4.8 mg (5.0 peptoid 14 and 29.22 mg (0.5 mmol) NaCl wereprepared. The following techniques were performed identically on 0.5 mMethynylferrocene (50 mM NaCl), 0.5 mM 13 (50 mM NaCl) or 0.5 mM 14 (50mM NaCl). Approximately 4 mL of solution was transferred to anelectrochemical cell and subjected to cyclic voltammetry (CV)experiments. Cyclic voltammetry was conducted using a CH Instruments600A Electrochemical Analyzer and CV curves were generated usingsoftware developed by CH Instruments. Current (μA) versus Potential (V)was measured across a freshly polished CHI104 3 mm diameter glassycarbon disk working electrode (CH Instruments) with a Ag/AgCl (3 M KCl)reference electrode (CH Instruments) and a Pt wire reference electrode(scan rate=9.0 mV s⁻¹).

Results

Experiments were initiated with the solid-phase synthesis of a linearpeptoid dodecamer scaffold including three azidopropyl sidechainssite-specifically positioned in the oligomer sequence (FIG. 1; Scheme 1,Compound 1). Peptoid scaffolds were synthesized with high efficiency onRink Amide resin using standard “submonomer” synthesis protocols (Hornet al., Bioconjugate Chem., 2004, 15, 428). Azide-functionalizedsidechains were conveniently integrated as N-substituents in the peptoidsequence using 3-azido-1-aminopropane (Carboni et al., J. Org. Chem.,1993, 58, 3736) as a submonomer reagent. Following synthesis andcharacterization of peptoid 1, the present inventors successfullyconjugated phenyl propargyl ether to the three azide groups on thedodecamer scaffold. Trivalent conjugation was achieved by reacting 1with phenyl propargyl ether in the presence of CuI, ascorbic acid andN,N′-diisopropylethylamine (DIPEA) in DMF/pyridine (7/3 v/v) at roomtemperature for 18 hours. This reaction resulted in the formation of a1,2,3-triazole linkage between the peptoid scaffold and the side chainconjugates (FIG. 1; Scheme 1, Compound 2).

The present inventors then investigated whether triazole linkagesgenerated by click chemistry cycloadditions are compatible with peptoidchain extension (FIG. 1; Scheme 1, Compound 3). Following synthesis of2, the click chemistry reagents were washed from the solid-phase andtwelve complete peptoid monomer addition cycles were executed. In orderto allow for azide coupling, three propargyl sidechains wereincorporated into the 24-mer peptoid scaffold 3. Benzyl azide wasconjugated to the three alkyne groups in a second round of clickchemistry modification to generate 4. All products shown in FIG. 1;Scheme 1 were characterized and confirmed after each elongation andcycloaddition cycle using Reversed-Phase High Performance LiquidChromatography (RP-HPLC) and Liquid Chromatography/Mass Spectrometrysequencing techniques (LC/MS^(n)). Both azide and alkyne groups withinthe oligomer sequence were modified with equal efficiency (>95%conversion).

Using this approach, the present inventors successfully generatedmulti-functionalized peptoid dodecamers that had been extended andmodified through four sequential cycles of click chemistry (FIG. 2;Scheme 2). Peptoid trimers containing terminal azide functionalities(FIG. 2; Scheme 2, Compound 5) were synthesized on solid-phase support.Compound 6 was generated by reacting 5 with phenyl propargyl ether inthe presence of CuI, ascorbic acid and DIPEA in 2-butanol/DMF/pyridine(5/3/2 v/v/v) at ambient temperature for 18 hours. The click chemistryreagents were washed from 6 and three complete rounds of peptoid monomeraddition were conducted, generating peptoid hexamer 7. Benzyl azide wasallowed to react with 7 in a second cycle of click chemistry to affordpeptoid 8. The technique of sequential elongation and cycloaddition wasrepeated until peptoid dodecamers comprising four distinct side chainconjugates attached at sites specifically modified for incorporationwere synthesized. Standard submonomer extension of peptoids 8 and 10afforded peptoids 9 and 11, respectively. Peptoids 10 and 12 weresynthesized through the conjugation of4-ethynyl-1-fluoro-2-methylbenzene and 1-ethynyl-4-pentylbenzene ontopeptoids 9 and 11, respectively. FIG. 3 shows overlaid analyticalRP-HPLC spectra of crude intermediates 6, 8, 10 and product 12 followingcleavage from solid-phase support. All products shown in FIG. 2; Scheme2 were characterized and confirmed after each elongation andcycloaddition cycle using RP-HPLC and LC/MS² sequencing. All azide andalkyne-containing coupling partners in FIG. 2; Scheme 2 were conjugatedwith their respective alkyne and azide reactive sites on the oligomerscaffold with high efficiency (>95% conversion). The overall crudepurity of final product 12 was found to be >75% as evaluated by RP-HPLC(FIG. 3, Trace 12).

The feasibility of using the sequential click chemistry method tointegrate multiple constituents suitable for the development of peptoidsas biosensor platforms was also explored. This involved selection of asensor moiety and a bioactive ligand as groups for conjugation. Thereversible redox properties of ferrocene/ferrocenium have previouslybeen exploited for biosensor applications (Casas-Solvas et al., Org.Lett. 2004, 6, 3687; Forrow et al., Bioconjugate Chem., 2004, 15, 137;Zhang et al., J. Am. Chem. Soc., 2005, 127, 10590). Additionally,estradiol is a typical representative of a class of clinically importanthormone ligands, leading to the study of estradiol conjugates forbiomedical applications (Blizzard et al., Biorg. Med. Chem. Lett., 2005,15, 3912). Reports have shown that stable organometallic hormonepharmacophores can be generated using a 17α-(ferrocenylethynyl)estradiolcomplex (Osella et al., Helv. Chim. Acta, 2001, 84, 3289). Utilizing thesequential click chemistry method, peptoid 13 (FIG. 4) was generated asa prototype sensor platform in which ethynylferrocene and17α-ethynylestradiol were site-specifically positioned along theoligomer scaffold. Methoxyethyl groups were incorporated as thepredominant sidechain in 13 in order to increase overall molecularhydrophilicity and impart water solubility to a compound incorporatingtwo hydrophobic moieties.

To test the effect of triazole conjugation on ferrocene redoxproperties, compound 13 was purified to >96% as determined by RP-HPLCand the electrochemical behavior of ethynylferrocene and 13 werecompared using cyclic voltammetry (CV). Additionally, the influence ofthe estradiol group on the redox potential of the neighboring ferrocenemoiety was evaluated by comparing the electrochemical characteristics of13 with its azido-functionalized precursor 14. CV experiments werecarried out at room temperature using previously described methods(Casas-Solvas et al., Org. Lett. 2004, 6, 3687). CV was performed onsolutions of ethynylferrocene (0.5 mM), 13 (0.5 mM) or 14 (0.5 mM)prepared in water with NaCl (50 mM) as a supporting electrolyte, using aAg/AgCl (KCl 3 M) reference electrode, a freshly polished glassy carbonworking electrode, and a Pt wire counter electrode with a scan rate of9.0 mV s⁻¹. Cyclic voltammograms of ethynylferrocene and 13 showedreversible redox couples of ferrocene/ferrocenium, as shown in FIG. 5.The values of the formal redox potential)(E^(o)) and the half-peakpotential (E_(p/2)) of ethynylferrocene, 13 and 14 are shown in Table 1.As expected, the ferrocene core of 13 showed a significant decrease inredox potential when compared to ethynylferrocene. This is attributed tothe altered electronic environment established by the extendedconjugation of the ferrocene cyclopentadiene group with the 1,2,3triazole ring (Rose et al., Inorg. Chem. 1993, 32, 781). Interestingly,E^(o) and E_(p/2) values for 13 and 14 were very similar, indicatingthat the redox potential of the ferrocene group is not affected byconjugation of a proximal bulky sub stituent. Because it is desirable toretain a similar relative signal intensity between sensor molecules thatcontain a variety of bioactive ligands, it is advantageous that theelectrochemical properties of the conjugated ferrocene are notsubstantially diminished by the neighboring estradiol. Future studieswill investigate the development of electrochemical-based biosensors inwhich site-specifically positioned sensor groups are used to report achange in redox potential upon protein binding by suitably modifiedpeptoid oligomers (Plumb et al., Bioconjugate Chem., 2003, 14, 601).

TABLE 1 Table 1 Electrochemical Data by Cyclic Voltammetry^(a) Entry E°(V) E_(p/2) (V) Ethynylferrocene 0.512 0.525 13 0.442 0.459 14 0.4420.456 ^(a)Cyclic voltammetry experiments were conducted onethynylferrocene (0.5 mM), 13 (0.5 mM) or 14 (0.5 mM) in water with NaCl(50 mM) as supporting electrolyte, a glassy carbon working electrode,Ag/AgCl reference electrode and a Pt wire counter electrode with a scanrate at 9.0 mV s⁻¹.

Example II

The present invention is directed to a novel method for generatingcompounds for polyvalent display. In particular, the present inventorsdescribe an implementation that allows synthesis of relativelyinexpensive and biologically relevant hormone-dependent agents thatexhibit significant advantages over those previously described in theliterature. Peptidomimetic scaffolds outfitted, for example, withmultiple 17α-ethynylestradiol and ferrocene functionalities can begenerated with minimal effort and cost. See FIG. 6, wherein a generalmethod for synthesis of polyvalent selective hormone receptor cytotoxicagents is shown. Triazole linkages between the ligand and scaffold areformed in highly regiospecific, thermodynamically favorable conjugationreactions that occur at room temperature in organic or aqueous solvents.Furthermore, the scaffolds used are N-substituted glycine oligomers(peptoids), which may prove beneficial in vivo due to their enhancedresistance to proteolytic degradation.

The oligomers shown, for example, in FIG. 7 may be utilized as in vitroselective hormone receptor cytotoxic agents to inhibit cellproliferation in breast cancer cell lines. Such cell-based assays areuseful for evaluating the potential efficacy of the polyvalent selectivehormone receptor cytotoxic agents of the present invention. In oneembodiment, the selective hormone receptor cytotoxic agents may betested for therapeutic efficacy by contacting MCF-7/Her 2 breast cancercells with compounds 1-3 to determine 1) if these compounds bind toestrogen receptors of MCF-7/Her 2 breast cancer cells and 2) to measurecellular proliferation and/or cytotoxicity in the presence of compounds1-3 to determine if these compounds impart cellular toxicity uponbinding. It is predicted that displaying selective hormone receptoragonists in a multivalent arrangement will allow for increased bindingavidity of compounds 1-3, enhancing their overall selectivity andtherapeutic effect. Additionally, with respect to the polyvalent displayof ferrocene on compounds 2 and 3, cytotoxic effects can be evaluated asa function of local ferrocene concentration. The anti-proliferativeeffects of compounds 1-3 will be compared to existing selective estrogenmodulator therapies presently in use, such as ferrocifen.

The structures of the following ethynyl-steroid conjugates are presentedas exemplary bioconjugates that may be utilized in the generation ofhormone-dependent cytotoxic agents using the methods of the presentinvention.

Example III

Additional applications of the invention include the synthesis ofmolecules with high affinity for a variety of biological targets, whichexhibit such high affinity as a consequence of the polyvalent display ofselect conjugated binding groups on a linear oligomeric scaffold. Theterm polyvalency is used herein to refer to high-affinity bindingbetween surfaces wherein the affinity is conferred at least in part bythe binding of repeated epitopes. It is noteworthy that polyvalency is adefining characteristic of the surfaces of almost all invadingpathogens. In addition, other applications include highly potentpharmaceuticals, whose therapeutic activity is anticipated to beenhanced due to the multiple presentation of cytotoxic moieties.Important applications in the field of biosensors, diagnostic agents,and molecular imaging probes are also encompassed by the presentinvention.

Targeting Viral Pathogens:

Viral molecules, particularly those displayed on a viral capsid, areexcellent targets against which to design polyvalent linear oligomers ofthe present invention which comprise heterogeneous pendant groups.Binding pairs comprising viral “ligands” and the cell surface moietieswith which they interact are known for a variety of viruses and cellulartargets. Viruses can bind to almost all classes of molecules on cellularsurfaces, including: sugars (polyoma and orthomyxoviruses, for example,recognize sialyloligosaccharides); phosphatidyl lipids (vesicularstomatitis virus (VSV), for example, recognize phosphatidylserine andphosphatidylinositol); and proteins (HIV recognizes CD4; humanrhinovirus recognizes intercellular adhesion molecule-1, ICAM-1). For areview, see Mammen et al., Angew. Chem. Int. Ed., 1998, 37, 2754 andKiessling et al., Angew. Chem. Int. Ed. 2006, 45, 2348, which areincorporated herein by reference.

The influenza virus, for example, is known to attach to the surface ofbronchial epithelial cells via interaction between multiple trimers ofthe lectin hemagglutinin (HA), which is densely packed on the surface ofthe virus, and multiple moieties of N-acetylneuraminic acid [sialic acid(SA)], the terminal sugar on many glycoproteins. See Mammen et al.(supra). In view of the above, it is envisioned that the method of thepresent invention may be used to design polyvalent viralligand-dependent cytotoxicity agents. Such agents would comprise apendant group or bioconjugate designed to interact with, for example, HAon the influenza virus and a cytotoxic moiety, such as those describedherein and known in the art. Bioconjugates may be modeled to mimic thestructure of SA as it is presented as a terminal sugar.

Targeting Bacterial Pathogens:

A variety of binding pairs comprising bacterial proteins and cellsurface proteins are known in the art and discussed in Mammen et al.(supra). In general, bacteria bind either directly to a cell surfacemolecule or moiety, or to molecules in the extracellular matrix ofpreferred tissues. Moreover, bacterial molecules have been identifiedthat bind to both sugars and proteins. Uropathogenic E. coli strains,for example, are known to attach both directly and indirectly to thesurface of epithelial cells in the urethra and bladder via polyvalentinteractions. Several bacterial surface proteins have been identifiedthat confer this tissue specificity, including: P-fimbrae (containingprotein G) and type I fimbrae (containing the FimH adhesin). Thelectin-like protein G, which is localized on the tips of P-fimbrialfilaments of uropathogenic bacteria, adheres to multiple copies of theGal(a1,4)Gal (PK antigen) portion of a glycolipid expressed on thesurface of epithelial cells in the urinary tract, especially the kidney.Multiple copies of E. coli F-protein attach polyvalently to fibronectin,a soluble glycoprotein which can bind polyvalently to the surface ofepithelial cells. As a result of these polyvalent interactions, andpotentially others, the E. coli collect and proliferate in tissues ofthe urinary tract, wherein they can cause disease, such aspyelonephritis.

It is, therefore, envisioned that the method of the present inventionmay be used to design polyvalent bacterial ligand-dependent cytotoxicityagents. Such agents would comprise a pendant group or bioconjugatedesigned to interact with either bacterial protein G or F-protein, forexample, on bacterial cells and a cytotoxic moiety, such as thosedescribed herein and known in the art. Alternatively, an oligomer of thepresent invention may be synthesized that displays pendant groups thatinteract with bacterial protein G, pendant groups that interact withbacterial F-protein, and at least one conjugated cytotoxic moiety.Bioconjugates for protein G interactors may be modeled to mimic thestructure of the Gal(a1,4)Gal (PK antigen) portion of a glycolipidexpressed in the urinary tract. Bioconjugates for F-protein interactorsmay be modeled to mimic the structure of the F-protein binding sites onfibronectin.

Targeting Cell-Cell Interactions:

Under some circumstances, it is desirable to reduce or inhibitinteractions between cells. One such example involves platelet bindingto arterial endothelial cells which can contribute to thrombotic events.In accordance with the present invention, polyvalent linear oligomerscan be synthesized that comprise two or more pendant groups thatspecifically bind to thrombogenic vascular wall proteins. Suchpolyvalent linear oligomers can be used therapeutically to block acuteplatelet deposition at sites of vessel injury by molecularly maskingthrombogenic vascular wall proteins. Arterial injury can result from avariety of natural events and invasive procedures, includingangioplasty.

Potent Pharmaceuticals:

Polyvalent oligomers comprising a plurality of pendant groups, whereinthe conjugated pharmaceutical moieties include two or more such moietiesare also envisioned. The structures of several azidonucleosides arepresented herein as exemplary bioconjugates that may serve as potentpharmaceuticals, particularly when presented multiply on an oligomericscaffold.

The present invention also encompasses polyvalent oligomers comprising aplurality of pendant groups, wherein the pendant groups are antibioticmoieties comprising the active site(s) of a desired antibiotic orantibiotics. Such polyvalent antibiotics are anticipated to act aspotent antibiotics by virtue of their polyvalent presentation ofantibiotic moieties. Polyvalent oligomers comprising a plurality ofantibiotic moieties conjugated as pendant groups may comprise multiplecopies of antibiotic moieties of a single antibiotic or multiple copiesof antibiotic moieties of different antibiotics. Polyvalent oligomerscomprising multiple copies of antibiotic moieties of a single antibioticare anticipated to exhibit enhanced potency. Polyvalent oligomerscomprising multiple copies of antibiotic moieties of differentantibiotics are likely to exhibit both enhanced potency and an expandedspectrum of activity. As such, polyvalent oligomers of the inventioncomprising a plurality of antibiotic moieties conjugated as pendantgroups may prove to be efficacious at lower doses as compared to thoserequired for standard “monovalent” antibiotics due to their multivalencywhich, in turn, enhances potency.

Biosensors, Diagnostic Agents, and Molecular Imaging Probes: The methodsof the present invention are also well suited to the synthesis of novelpolyvalent oligomers comprising more than one type of pendant group,wherein the conjugated pendant group confers upon the oligomer theability to function as a detector for the presence and/or activity of aparticular molecule. Such functional groups include, but are not limitedto: fluorophores, chelates, radioisotopes, affinity tags, and numerousother groups known to skilled practitioners (G. T. Hermanson,Bioconjugate Techniques, Academic Press: San Diego, Calif., 1996). Toenhance the versatility of such polyvalent oligomers, differentfunctional groups that interact with each other in a detectable mannercan be conjugated to the same oligomeric backbone. Such groups mayconfer a detectable signal when they are, for example, brought within acertain proximity, perhaps as a consequence of a conformational changein a molecule to which the polyvalent oligomer has bound.

Example IV

In another aspect, the method of the present invention may be used tosynthesize polyvalent molecules comprising multiple copies of identicalconjugated pendant groups on a linear oligomeric scaffold. Exemplarypendant groups for conjugation to a linear oligomeric scaffold of thepresent invention are presented herein above. A skilled practitionerwould, however, be capable of envisioning other pendant groups ofutility based on the teaching of the present invention. Such polyvalentmolecules are anticipated to exhibit enhanced properties as a result oftheir polyvalent nature. With respect to polyvalent oligomers thatpresent multiple copies of therapeutic moieties, such polyvalentmolecules are likely to display dramatically improved therapeuticefficacy relative to their “monovalent” counterparts.

As described herein, an efficient protocol to effect multi-siteconjugation reactions to oligomers on solid-phase support is presented.Sequence-specific N-sub stituted glycine “oligopeptoids” were utilizedas substrates for azide-alkyne cycloaddition reactions. Diverse groups,including nucleobases and fluorophores, were conjugated at up to 6positions on these peptoid sidechains with yields ranging from 88% to96%. This strategy is broadly applicable for generating polyvalentdisplays on oligomeric backbones and allows precise control of spacingbetween pendant groups attached thereto.

As indicated above, the strategy entails the synthesis of linear peptoidsequences including multiple reactive groups at specific sidechainpositions (FIGS. 10 and 11). Peptoids were conveniently synthesized bystandard “submonomer” automated protocols. Azide or alkyne functionalgroups were readily incorporated using 1-azido-3-aminopropane orpropargylamine as submonomer reagents, affording oligomers 1a-2c onsolid phase support. Corresponding alkyne or azide partners,respectively, were conjugated to these selectively reactive sites on thepeptoid scaffolds (FIG. 12).

To demonstrate the broad utility of this reaction, diverse couplingpartners were employed (Table 2). PRODAN, or6-propionyl-2-(dimethylamino)naphthalene is a solvatochromic fluorophorethat has proven useful as probe in biological systems. The presentinventors conjugated the 6-azido-acetyl analog 7 to three sites on thepeptoid scaffold 2a and synthesized a trivalent peptoid fluorophore 8(Table 2, entry 1).

A trivalent peptoid-nucleoside conjugate 10 was similarly synthesizedusing azidothymidine 9 (Table 2, entry 2). Nucleobase conjugation topeptoid scaffolds at multiple residues offers a new type of syntheticoligomer similar to peptide nucleic acids for use as novel probes anddiagnostic agents.

Peptoid trimers 11 to three positions on a helical peptoid^(2b) octamer2c, constructing a trivalent display 12 (Table 2, entry 3). This complexmolecule was generated by a convergent “one-step” reaction,demonstrating the capability of click chemistry to efficiently generateelaborate branched architectures.

TABLE 2 Synthesis of Trivalent Conjugates on Peptoid Scaffold entrycoupling partners scaffolds 1

  7 2a 2

  9 2a 3

  11 2c entry product conditions^(a) 1

  8 A 2

  10 A 3

  12 B

Prior to the present invention, sodium ascorbate ortris(carboxyethyl)phosphine were used for in situ reduction of Cu(II)salts in aqueous systems. As indicated herein, the present inventorsdemonstrate that Cu(I) salts can be used directly without complicationsin organic solvent systems, when stabilized by ascorbate in the presenceof DIPEA. Reactions are conducted without: the formation of undesiredby-products, the necessity for prior preparation of Cu(I) ligands, orthe need for rigorous exclusion of oxygen. Indeed, the present inventorshave successfully utilized ascorbic acid in a variety of organic solventsystems including pyridine, DMF and alcohols, underscoring thecompatibility of this method with diverse solid phase resins. Thus, atlow cost and remarkable convenience, these procedures can beincorporated into common solid phase organic synthesis protocols,potentially allowing seamless utilization in automated synthesizers.

As alluded to herein above, the polyvalent display of therapeuticmoieties as conjugates displayed on oligomeric backbones offers thepotential for the generation of super potent polyvalent therapeuticand/or prophylactic pharmaceuticals. Such polyvalent pharmaceuticals maybe administered to subjects in need thereof alone or in the form of acomposition further comprising a physiologically acceptable excipient. Askilled practitioner would be able to identify subjects for whom suchadministration would confer benefit and would, moreover, be able todetermine suitable doses for administration based on a number ofparameters (e.g., disease afflicting the patient, weight, age andcondition of the patient).

Exemplary polyvalent therapeutic oligomers of the present inventioninclude estradiol-containing peptoids, which are shown below as mono-,di-, trivalent conjugates. A hexavalent estradiol-peptoid conjugate isshown in FIG. 13B.

The following structure shows an estradiol-peptoid conjugate comprisinga poly-Arg tail:

Example V Polyvalent Display of Peptides on a Peptoid Scaffold

FIG. 8 (Scheme 4) shows a hypothetical solid phase synthesis of apeptide oligomer in which the N-terminus amine has been converted to anazido group. The first steps show the solid-phase peptide synthesis(SPPS) of the primary peptide sequence. In step “a”, F-moc deprotectionis performed with 20% piperidine (base) in N-methyl-2-pyrrolidone (NMP).This step is followed by azide functionalization (step “b”) by treatingthe resin-bound oligomer with trifluoromethylsulfonyl azide (CF3SO2-N3)in the presence of copper sulfate (CuSO4) in dichloromethane/methanol(9/1 v/v) at ambient temperature for 16 hrs. Finally, in steps “c” and“d”, respectively, the peptide is cleaved from the resin withtrifluoroacetic acid (TFA) (50% in DCM) and washed with 0.02Mdiethyldithiocarbamic acid sodium salt. See also Rijkers et al., 2002,Tet. Letters 43, 3657.

Butynoic acid can also be coupled to the end of a growing peptidesequence using standard coupling procedures. This coupling step may beused to render the peptide chain “clickable.” Such coupling proceduresmay be used to advantage in generating peptoid-peptide hybrids, forexample. A structure of butynoic acid is shown below:

FIG. 9 (Scheme 5) outlines the click chemistry procedure of the presentinvention whereby azido peptides may be polyvalently displayed on apeptidomimetic scaffold. Equivalents are to be optimized throughexperimental results, but a good starting point is envisioned to utilize40 eq Cu, 20 eq ascorbic acid, and 50 eq DIPEA. The cleavage shown instep 2 is achieved via treatment of the resin with 95% TFA in water for10 min.

Example VI Multivalent Estradiol-Peptidomimetic Conjugates

Using the technology described herein, the present inventors havegenerated a versatile new class of multivalent hormone conjugates forselectively modulating the activity of estrogen receptors (ER), whichare among the most important therapeutic targets in breast cancer. SeeFIG. 13, wherein one of the multivalent estradiol-peptidomimeticconjugates, a hexavalent peptoid estradiol conjugate is depicted.

Estrogen hormones such as 17β-estradiol (E2) are known to play acritical role in the development and progression of many human breastcancers (Thomas et al., Curr. Canc. Drug Tar., 2004, 4, 483). E2 is anatural ligand for ERα and ERβ, which have classically been described asnuclear receptors. In this role, ligand-activated ER functions via“genomic” pathways following nuclear localization by altering the extentof target gene transcription. In addition, estrogens are now also knownto exhibit rapid effects via membrane-associated receptors that cantrigger a set of “nongenomic” pathways, such as kinase cascades(Acconcia et al., Cancer Letters, 2006, 238, 1). The relationshipbetween the genomic and non-genomic pathways is poorly understood.Further elucidation of the varying mechanisms of estrogen action is apriority for enhancing knowledge of breast cancer pathogenesis andmolecular pharmacology. One promising approach is to develop estrogenicligands that are capable of selectively activating a specific pathway.Macromolecular forms of estradiol, such as E2 conjugated to BSA protein,for example, have been prepared in studies that aim to activate themembrane-associated ER exclusively (Stevis et al., Endocrinology, 1999,140, 5455). The stability and activity of estrogen-protein conjugates ishighly variable, however, underscoring the need for optimizedmacromolecular estrogen conjugates (Harrington et al., Mol. Endocrinol.,2006, 20, 491).

To address this need, the present inventors have developed biomimeticpolymers as a scaffold for a family of compounds that can be tailored toselectively modulate genomic or nongenomic ER pathways. In accordancewith the present invention, the inventors have used “click chemistry”reactions to generate several compounds bearing a precise multivalentdisplay of the ER ligand on a monodisperse water-soluble scaffold.Indeed, this approach may be used to generate a library of compoundsbearing a precise multivalent display of ER ligands on a monodispersewater-soluble scaffold. See FIG. 13A (n=2 to 10), for schematic of suchmultivalent estradiol conjugates. FIG. 14B depicts the chemicalstructure of a hexavalent peptoid estradiol conjugate, the activity ofwhich has been tested and quantitated as described below.

Materials and Methods

Mammalian Cell Extracts: MCF-7 cells were grown to ˜80% confluence in10-cm culture dishes and harvested in 5 ml ice cold phosphate bufferedsaline (PBS). Cell suspensions were centrifuged and the pellets weresnap frozen on dry ice to lyse the cells. Cell pellets were resuspendedin 500 μl freshly prepared receptor buffer (50 mM NaCl, 10 mM Tris pH7.5, 1 mM EDTA, 1 mM DTT (dithiothreitol), 15 mM MgCl₂, 20 mM sodiummolybdate, 20% glycerol, 1 protease inhibitor), and incubated on ice for10 min. The lysates were centrifuged for 20 min at 15,000×g for 20 minat 4° C. The protein concentration of each supernatant was assayed usinga Bio-Rad protein assay and was typically between 1-2 μg/μl.

Competitive Binding Assays: Cell extracts were incubated with 1.0×10⁻⁸ Mradiolabeled ligand (³H-Estradiol) in the presence or absence ofincreasing concentrations of unlabeled ligand in a final volume of 100μl of receptor buffer. Following 18 hr incubation at 4° C., cellextracts were mixed with an equal volume of a 10 mg/ml activatedcharcoal suspended in receptor buffer and incubated on ice for 10 min.The activated charcoal/cell extract slurry was centrifuged at 12,000×gfor 3 minutes. 180 μl cell extract supernatant was added to 2 mlscintillation fluid and ³H decay was counted for 3 minutes on ascintillation counter. Binding was computed as scintillation counts perminute (cpm) in the absence of unlabeled ligand minus cpm in thepresence of unlabeled ligand. Non-linear regression analyses wereperformed using GraphPad Prizm® software.

Luciferase Assays: Human Embryonic Kidney (HEK) 293T cells were platedonto 24 well plates coated with poly-D lysine and grown to ˜70%confluence in yellow Dulbecco's Modified Eagles Media (DMEM) containing5% charcoal-stripped fetal bovine serum (CS-FBS). Cells were stablytransfected with xETL, pCMV(LacZ), Bluescript and either pcDNA3(wtER) orpcDNA3(Vo) plasmids. Cells were allowed to recover in yellow DMEMcontaining 5% CS-FBS for 6 hrs and then serum starved for 12 hrs inyellow DMEM containing 0% CS-FBS before ligand treatment. Cells werethen treated with 100 nM final concentration of respective ligand inEtOH for 18 hrs before harvesting. After harvesting, the cells wereincubated in Promega™ lysis buffer for 30 min at room temperature. Celllysates were plated in duplicate on 96-well plates, reacted withluciferase assay buffer (Promega), and read on an Lmax plate reader. Totest transfection efficiency and for normalization, cell lysates wereplated in duplicate on 96-well plates, reacted with LacZ buffer, andread on a Molecular Biosystems plate reader to assay β-gal activity.Data were processed using the Microsoft Excel spreadsheet program.

Results

In accordance with the present invention, monovalent, divalent,trivalent, and hexavalent peptoid estradiol conjugates were synthesized.MCF-7 cell extracts were prepared from logarithmically growing MCF-7cells and binding of monovalent, divalent, trivalent, and hexavalentpeptoid estradiol conjugates to purified ER was evaluated by competitiveradiometric assay as described herein. As shown in FIG. 14, increasingthe valency of estradiol presentation is correlated with an increase inavidity of binding between the peptoid estradiol conjugates and ERpresent in the cellular extracts. Binding avidity of the peptoidestradiol conjugates is compared to the positive control of17β-estradiol (E2). The inset of FIG. 14 shows a saturation curve ofbinding activity for the trivalent peptoid estradiol conjugate.

The peptoid estradiol conjugates were also assessed with respect totheir ability to activate genomic pathways downstream of the estrogenreceptor (ER). Briefly, ER+(wtER) and ER− (Vo) human embryonic kidney(HEK) 293T cells were stably transfected with plasmids containingER-responsive luciferase reporter genes. These transfected cell lineswere used as cell based assays to measure transcriptional activationtriggered by ER engagement. Cells were treated for 18 hrs with 100 nMconcentrations of the indicated ligand. Luciferase reporter assays wereused to assess activation. As shown in FIG. 15, all of the peptoidestradiol conjugates stimulated an increase in transcription of ERresponsive luciferase reporter genes as measured by an increase inluciferase activity.

One of skill in the art would also appreciate that genomic mechanisms ofER action may also be evaluated using other protocols, such as thosethat call for reverse transcriptase polymerase chain reaction (RT-PCR)amplification of transcripts of genes known to be regulated on atranscriptional level by ER engagement.

Similar cell based systems may also be used to measure activation ofnon-genomic signaling pathways. For such applications, cell lysateswould be prepared for subsequent Western Blot analysis of, for example,phosphorylated targets. Fluorescently labeled conjugates may also beused to visualize their sub-cellular localization.

As indicated above, the present method may be used to generate a libraryof compounds bearing a precise multivalent display of ER ligands on amonodisperse water-soluble scaffold. Moreover, new synthetic routes willalso be established for the precise multivalent display of estradiolconjugates. Diverse sequences so generated will be screened to identifyspecies that are excluded from trafficking to the nucleus and are thuscapable of selectively activating membrane-associated ER signaling. Thebiological activity of such compounds will be rigorously evaluated inhuman breast cancer cell lines such as, for example, ER(+) MCF-7 and inER(−) control cell lines. Large estradiol conjugates are anticipated toevoke a specific subset of ER-mediated pathways within the cell.Although not limiting with respect to the novel macromolecular estradiolconjugates of the present invention, it has been observed thatincreasing the size and charge of estrogen conjugates mitigates theircapability to traffic to the nucleus (Patch et al., Curr. Opin. Chem.Biol., 2002, 6, 872). In addition, the binding of macromolecularestradiol conjugates to the ER may sterically block the association ofprotein co-activators or occlude functional interactions with estrogenresponse elements on target gene promoters. As a result, genomicresponses to ER signaling may be selectively abrogated. Correspondingly,these effects may facilitate more rigorous examination of nongenomicactions of estrogen.

Novel macromolecular estradiol conjugates of the present invention maybe found that curtail the ability of ligand-activated ER to evokegenomic responses, thus yielding a family of lead compounds for improvedhormone replacement therapy and for the inhibition of hormone-responsivebreast cancer proliferation. The significance of such research will bethe discovery of a new family of molecules to study and control thefunction of estrogen hormone receptors, which play a crucial role in thedevelopment of many breast cancers.

The present method may, for example, be used to generatepeptoid-ethisterone conjugates such as those shown in FIG. 16. Compoundsin FIG. 16 show mono-, di- and trivalent display of the progesteronereceptor ligand ethisterone. Briefly, azide-containing peptoid scaffoldswere synthesized on solid-phase support as described herein. Ethisteronewas efficiently conjugated to the molecular scaffold using the CuCAACreaction, resulting in a 1,2,3 triazole linkage between the receptorligand and the peptoid scaffold. Compounds shown here may be used tostudy agonist or antagonist effects with a diverse array of sex steroidhormone receptors.

Similar approaches may be applied to the development of macromolecularconjugates designed to target prostate cancer cells and other hormoneresponsive tumors. In accordance with the present invention, multivalentandrogen conjugates, for example, may be synthesized and tested in cellbased assays, which are known to skilled practitioners, to evaluatetheir potential as diagnostic and/or therapeutic compounds. Otherhormone responsive tumors include, without limitation, kidney andpituitary tumors.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1-10. (canceled)
 11. A polyvalent linear peptoid comprising a pluralityof heterogeneous backbone-attached pendant groups, wherein each of theheterogeneous backbone-attached pendant groups is attached to thebackbone of the linear peptoid via a triazole linkage group. 12-21.(canceled)
 22. The polyvalent linear peptoid of claim 11, wherein thepeptoid comprises between 2-1,000 monomers.
 23. The polyvalent linearpeptoid of claim 11, wherein the peptoid comprises between 2-100monomers.
 24. The polyvalent linear peptoid of claim 11, wherein thepeptoid comprises between 2-25 monomers.
 25. The polyvalent linearpeptoid of claim 11, wherein the peptoid is di-valent, tri-valent,tetra-valent, penta-valent, hexa-valent, hepta-valent, octa-valent,nona-valent, and deca-valent.
 26. The polyvalent linear peptoid of claim11, wherein the backbone-attached pendant groups comprise hormonereceptor ligands, cell surface receptor ligands, tumor specific antigenligands, cytotoxic agents, pharmaceutical moieties, fluorophores,chelates, radioisotopes, affinity tags, or antibiotic moieties.
 27. Thepolyvalent linear peptoid of claim 11, wherein the triazole linkagegroup is formed by transformation of ethynyl or azido of the pendantgroup.
 28. The polyvalent linear peptoid of claim 27, wherein thetransformation of ethynyl or azido of the pendant group occurs viaazide-alkyne [3+2]-cycloaddition conjugation.
 29. The polyvalent linearpeptoid of claim 11, wherein the backbone-attached pendant groupscomprise steroid hormone receptor ligands.
 30. The polyvalent linearpeptoid of claim 11, wherein the backbone-attached pendant groupscomprise hormone receptor ligands, and the hormone receptor ligand is17a-ethynyltestosterone, 17a-ethynylestrdiol, norgestimate,17a-proynylestradiol, or 17a-butynylestradiol:


31. The polyvalent linear peptoid of claim 11, wherein thebackbone-attached pendant groups comprise hormone receptor ligands, andeach hormone receptor ligand is attached to the backbone via a triazolelinkage which is formed through the transformation of the triple bond ofthe hormone receptor ligand.
 32. The polyvalent linear peptoid of claim11, wherein the backbone-attached pendant groups comprise hormonereceptor ligands; each hormone receptor ligand is attached to thebackbone via a triazole linkage which is formed via azide-alkyne[3+2]-cycloaddition conjugation.
 33. The polyvalent linear peptoid ofclaim 11, wherein the pendant groups are selected from phenyl propargylether, (4-pentylphenyl)ethyne, (4-fluoro-3-methylphenyl)ethyne,3-phenylpropyne, and ethynylferrocene; and each pendant group isattached to the backbone via a triazole linkage which is formed throughthe transformation of the triple bond of the pendant group.
 34. Thepolyvalent linear peptoid of claim 11, wherein the pendant groups areselected from phenyl propargyl ether, (4-pentylphenyl)ethyne,(4-fluoro-3-methylphenyl)ethyne, 3-phenylpropyne, and ethynylferrocene;and each pendant group is attached to the backbone via a triazolelinkage which is formed via azide-alkyne [3+2]-cycloadditionconjugation.
 35. The polyvalent linear peptoid of claim 11, wherein thependant groups are selected from

and each pendant group is attached to the backbone via a triazolelinkage which is formed through the transformation of the azido group ofthe pendant group.
 36. The polyvalent linear peptoid of claim 11,wherein the backbone comprises N-substituted glycine monomers.
 37. Thepolyvalent linear peptoid of claim 11, wherein the backbone comprisesN-substituted glycine monomers, and the substitution on the N ismethoxyethyl, aminopropyl, aminobutyl, imidazoethyl, imidazomethyl,benzyl, phenethyl, naphthylmethyl, or guanidinopropyl.
 38. Thepolyvalent linear peptoid of claim 11, wherein the polyvalent linearpeptoid is


39. The polyvalent linear peptoid of claim 11, wherein the polyvalentlinear peptoid is


40. The polyvalent linear peptoid of claim 11, wherein the polyvalentlinear peptoid is


41. A composition comprising the polyvalent linear peptoid of claim 11and a physiologically acceptable excipient or a carrier.